Infection by Mycobacterium tuberculosis (Mtb) is currently a major global health problem, resulting in 2 million deaths from tuberculosis each year. Characterization of the gene regulatory mechanisms in mycobacteria will increase our understanding of the bacterial factors necessary to establish a successful infection. The recent discovery of the presence of up to 15 different adenylate cyclases (ACs) in the Mtb genome suggests that cAMP-mediated gene regulation may be an important, yet overlooked, global regulatory mechanism in mycobacteria. The long-term goal of this project is to gain insights into the molecular and cellular cAMP-mediated responses of Mycobacterium smegmatis, with the belief that these insights will also shed light on the cAMP-regulated responses of Mtb. A majority of the Mtb ACs and cAMP- binding proteins have homologs in the M. smegmatis genome, indicating that aspects of cAMP regulation may be conserved among all mycobacteria. The specific aims of the proposed project are: Aim 1: characterize the regulation of mycobacterial ACs by examining the expression of Mtb cyclases and their M. smegmatis homologs using a combination of LacZ:reporter fusions, RT-PCR and protein detection. AC regulation will be studied in various environmental conditions, with a focus on conditions faced by Mtb during macrophage infections and times of latency;Aim 2: identify key proteins necessary for the expression and regulation of mycobacterial ACs by screening a M. smegmatis mutant library for changes in wild-type gene expression. Mutant libraries will be constructed utilizing a transposon mutagenesis system delivered by mycobacteriophage. Aim 3: identify proteins involved in mycobacterial cAMP secretion by screening a M. smegmatis mutant library for changes in the level of cAMP secretion in different environments;Aim 4: develop a successful undergraduate research program at Ferrum College to expose students early in their careers to novel, exciting scientific research. As an AREA grant these studies will heavily involve the work of undergraduates who will be responsible for performing many of the experiments and will be included in information dissemination by presenting posters at scientific meetings and being co-authors on related publications. Utilizing M. smegmatis as an Mtb surrogate allows undergraduates to gain experience in key laboratory techniques while studying an important public health problem in a safe and productive environment. This work will explore a previously understudied gene regulatory network in mycobacteria and provide a foundation for future studies using M. smegmatis to model the cAMP-regulatory networks of Mtb. PUBLIC HEALTH RELEVANCE: Tuberculosis is a major global health problem, and new intervention strategies are desperately needed to combat this disease. This work will utilize a nonpathogenic mycobacterial species to explore a previously understudied global regulatory system in mycobacteria, with the potential to relate information gained to pathogenic Mtb. Characterization of gene regulatory mechanisms in Mtb will increase our understanding how TB bacteria establish a successful tuberculosis infection and help to identify potential targets for TB pharmaceuticals or vaccines.